O-6-ALKYLDEOXYGUANOSINE DETECTION BY P-32-POSTLABELING AND NUCLEOTIDE CHROMATOGRAPHIC ANALYSIS

Abstract

The 32P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O6-alkyl-2''-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3''-monophosphates and the O6-alkyldeoxyguanosine-3''-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3''-monophosphates (e.g., O6-alkyldeoxyguanosine-3''-monophosphates) were then converted to labeled deoxyribonucleotide-[5''-32P]monophosphates by 32P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O6-methyl- and O6-ethyl-2''-deoxyguanosine-3''-monophosphate nucleotides, and the respective 5''-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O6-methyl- and O6-ethyl-2''-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2''-deoxyguanosine residue per 104 and 107 2''-deoxyguanosines. The limit of detection was less than one O6-alkyl-2''-deoxyguanosine in 107 2''-deoxyguanosine residues in a sample size of 100 .mu.g of DNA, i.e., approximately 10 pg of adduct. The quantitation of O6-methyl-2''-deoxyguanosine in the liver DNAs of rats treated with [14C-Me]N-nitrosodimethylamine compared well with values obtained by both 14C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these 32P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.